Cox M A, Jenh C H, Gonsiorek W, Fine J, Narula S K, Zavodny P J, Hipkin R W
Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Mol Pharmacol. 2001 Apr;59(4):707-15. doi: 10.1124/mol.59.4.707.
The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-gamma), and I-TAC (interferon-inducible T cell alpha chemoattractant) attract lymphocytes through activation of CXCR3. In the studies presented here, we examined interaction of these chemokines with human CXCR3 expressed in recombinant cells and human peripheral blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating [(35)S]GTP gamma S binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. (125)I-IP-10 and (125)I-I-TAC bound hCXCR3 with high affinity, although the (125)I-I-TAC B(max) value in saturation bindings was 7- to 13-fold higher than that measured with (125)I-IP-10. Coincubation with unlabeled chemokines decreased (125)I-IP-10 binding with a single discernible affinity. However, with (125)I-I-TAC, competition with IP-10 or MIG was incomplete, and multiple binding affinities were evident. Moreover, in contrast to I-TAC, IP-10 and MIG binding IC(50) values did not increase predictably with increased (125)I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated (125)I-IP-10 binding but only decreased (125)I-I-TAC binding 30 to 80%, indicating that unlike IP-10, I-TAC binds with high affinity to uncoupled (R) and coupled (R*) hCXCR3. To examine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibody on I-TAC- and IP-10-stimulated [(35)S]GTP gamma S binding. The antibody attenuated [(35)S]GTP gamma S binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently to R*. Moreover, increased occupancy of R* with a >75-fold increase in (125)I -IP-10 concentration did not increase the I-TAC binding IC(50) value, and I-TAC increased the dissociation rate of (125)I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.
人CXC趋化因子IP-10（10 kDa干扰素诱导蛋白）、MIG（人γ干扰素诱导的单核因子）和I-TAC（干扰素诱导的T细胞α趋化因子）通过激活CXCR3吸引淋巴细胞。在本文介绍的研究中，我们检测了这些趋化因子与重组细胞和人外周血淋巴细胞（PBL）中表达的人CXCR3的相互作用。IP-10、MIG和I-TAC是刺激重组细胞膜和PBL膜中[(35)S]GTPγS结合的激动剂，但在无hCXCR3表达时无作用。(125)I-IP-10和(125)I-I-TAC与hCXCR3高亲和力结合，尽管饱和结合中(125)I-I-TAC的B(max)值比(125)I-IP-10测得的值高7至13倍。与未标记的趋化因子共孵育以单一可识别的亲和力降低了(125)I-IP-10结合。然而，对于(125)I-I-TAC，与IP-10或MIG的竞争不完全，且有明显的多种结合亲和力。此外，与I-TAC不同，在竞争结合中，IP-10和MIG的结合IC(50)值不会随着(125)I-I-TAC浓度增加而可预测地增加，表明这些趋化因子是非竞争性（即别构）配体。hCXCR3的解偶联消除了(125)I-IP-10结合，但仅使(125)I-I-TAC结合降低30%至80%，表明与IP-10不同，I-TAC与未偶联的（R）和偶联的（R*）hCXCR3高亲和力结合。为了检测趋化因子与R的结合，我们测试了抗hCXCR3抗体对I-TAC和IP-10刺激的[(35)S]GTPγS结合的影响。该抗体减弱了对IP-10而非I-TAC的[(35)S]GTPγS结合反应，表明这两种趋化因子与R的结合方式不同。此外，(125)I -IP-10浓度增加>75倍导致R占有率增加，但未增加I-TAC的结合IC(50)值，且I-TAC增加了(125)I-IP-10的解离速率。根据这些数据，我们得出结论，IP-10和I-TAC与hCXCR3的R状态的结合是别构的。
